Ultra-pure thrombin preparation

ABSTRACT

An ultra-pure, clear thrombin solution having a high specific activity is described as well as a method of manufacture.

This is a divisional application of U.S. Ser. No. 07/973,014, filed Nov.6, 1992, now U.S. Pat. No. 5,281,528, which is a divisional applicationof U.S. Ser. No. 07/657,427, filed Feb. 22, 1991, now pending; which isa continuation-in-part application of U.S. Ser. No. 07/452,174, filedDec. 18, 1989, now abandoned.

BACKGROUND

Thrombin, a proteolytic enzyme, is essential for hemostasis. It is aprinciple reagent in the formation of blood clots via fibrin production.Due to its effectiveness as a clotting aid, thrombin and itspreparations are useful during surgical procedures to control bleeding.While dry thrombin is available, liquid preparations are generallypreferred due to handling and time considerations.

Until now, there have been no highly stable, clear liquid thrombinpreparations which are both storage stable and ready for use duringsurgery. This is because thrombin, when dissolved in water or saline,rapidly loses its activity due to denaturation and autolysis of thethrombin protein.

THE INVENTION

The present invention is directed to a novel modification of a processfor the preparation of thrombin of ultra-pure quality in solution. Thissolution is completely clear and free of turbidity, and has acharacteristic of high clotting activity, less inactive protein, and ahigh specific activity, more than any thrombin product hereto described.

The novelty of the present process, which achieves the goal of a clearthrombin solution, is the unique combination of a whole host of steps ina particular sequence.

First of all, the common thrombin, in circulating blood, exists in aninactive form called prothrombin and a factor called thromboplastin isrequired in order to convert prothrombin to thrombin. The presentinvention does not involve the usual use of bovine lung extract as asource of thromboplastin but uses an isolated, highly purifiedthromboplastin, as described hereinafter. This process eliminates asignificant amount of contaminating proteins which are the probablesource of impurities and turbidity often seen in a final product.

The prothrombin to thrombin conversion mixture, following the usualcentrifugation step, is passed through an anion exchange chromatographycolumn, affording an eluted material which is still turbid. Thismaterial is then, in the present invention, frozen and then thawed,followed by centrifugation to remove most of the turbidity. Removal ofthis turbidity improves the solution flow through the second stagecation-exchange column chromatography procedure.

Following the second passage of the solution through a cation exchanger,the improvement in this step comprises eluting the material through thiscolumn by a normal flow from top to bottom with a salt gradient ratherthan a standard sodium chloride solution.

The results of these modifications have provided the isolation of awater-clear, ultra-pure thrombin, the specific activity being muchsuperior to any product available on the market, as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Comparison Between Thrombostat ®, Thrombinar ®,                       and Ultra-Pure Thrombin                                                                                      Ultra-                                                  Thrombostat ®.sup.a                                                                 Thrombinar ®.sup.b                                                                    Pure                                           ______________________________________                                        Clotting activity.sup.c                                                                  2164        1372        7820                                       (U/mL)                                                                        Protein (mg/mL)                                                                          11.56       0.82        0.82                                       Specific activity                                                                        187         1663        9500                                       (U/mg)                                                                        ______________________________________                                         .sup.a Parke-Davis                                                            .sup.b Armour                                                                 .sup.c Determined by a modified NIH method hereinafter described.        

Accordingly, the present invention concerns, in its broadest aspects:

I. An ultra-pure, clear, colorless thrombin solution having specificactivity from 4000 to 11,000 Units/mg protein.

II. An ultra-pure, clear, colorless thrombin solution prepared byreacting prothrombin with purified thromboplastin and treating theresulting thrombin, after centrifugation, by eluting the supernatantthrough an anion-exchange agarose column; freezing, then thawing up toabout 25° C., the desired eluant fractions; centrifuging and eluting thesupernatant through a cation-exchange agarose column with a saltgradient in a buffer.

III. A process for preparing an ultra-pure, clear, colorless thrombinsolution comprising:

reacting prothrombin with purified thromboplastin; centrifuging thesuspension; eluting the supernatant through an anion-exchange agarosecolumn with buffer; freezing then thawing up to about 25° C. the desiredeluant fractions; centrifuging and eluting the supernatant through acation-exchange agarose column with a salt gradient in a buffer.

ADVANTAGES

The thrombin compositions and methods of the invention have severaladvantages over conventional preparations and methods for assisting inblood clotting.

Unlike powdered preparations, the compositions of the instant inventionrequire no reconstitution prior to use. Thus, measuring, mixing,sterilizing, etc. of one or more component(s) or container(s) are notimportant considerations. The instant clear preparations can be usedwithout preparation before final use because of the absence of particleswhich cause the turbidity in former liquid preparations.

Furthermore, the stability of the instant thrombin-containing materialsis such that the need for stock inventories and/or rotation of productsis largely eliminated. Unlike most saline or water-solutions ofthrombin, which are stable for only about one week at 4° C., the instantpreparations are designed to be stable at normal refrigerationtemperatures (i.e., about 4° C.) and at room temperature (i.e., about25° C.) for 6 months or more.

It is known that high concentrations of glycerol, sucrose, and otherpolyols can stabilize proteins in solution. In the case of thrombin, itis known that a glycerol concentration of 67% can greatly stabilize a1,000 μ/mL thrombin solution. However, use of high glycerolconcentrations is not practical in the large scale manufacture of asterile thrombin solution because of the high viscosity of such apreparation. The instant compositions, which may contain 30% or less, ofglycerol avoid these problems.

Other advantages and aspects of the invention will become apparent froma consideration of the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the purification of thrombin by way of theprofile of elution of the CM-Sepharose® column using the salt gradientof 0.1 to 1.0M sodium chloride solution as described in the example.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Isolation of Thromboplastin From Lung Tissue

Bovine/veal lung tissue is trimmed to remove fatty tissue, cut intosmall pieces, and homogenized in a tissue homogenizer with 25-50 mMphosphate buffer pH 6.2-6.7, preferably pH 6.5 containing 0.5-1.0%polysorbate 80. The surfactant aids in the extraction of the enzyme.Other nonionic surfactants can be used, e.g., fatty acid esters ofpolyoxyethylene sorbitan, e.g., Tween®, ICI; polyoxyalkylyne fatty acidesters, e.g., MYRJ®, ICI; polyoxyethylene fatty ethers, e.g., Brij®,ICI; polyoxypropylene-polyethylene ethers, e.g., Pluronic®, BASF;sucrose mono esters; and Triton X-100. The homogenate is centrifuged at12-14K RPM and the supernatant is collected. The pH of the supernatantis adjusted to pH 5.2-5.7, preferably pH 5.5 using drops of 10% aceticacid and centrifuged at 12-14K RPM. The supernatant from this step isloaded on a conventional cation exchange crosslinked agarose column,e.g., CM-Sepharose® (other cationic resins can be used, e.g.,CM-Sephadex®) and eluted with 25-50 mM phosphate buffer as describedabove. The eluate is monitored at 280 nM. The activity of thromboplastinin the eluate is also monitored indirectly by first convertingprothrombin to thrombin and the latter activity is then determined bymeasuring clotting activity using a fibrometer.

The initial fractions eluting from the column form part of the firstpeak which contains thromboplastin. Fractions eluting later which arepart of the second peak contain nonactive extraneous proteins. Thispurified thromboplastin solution is opalescent without any haze orturbidity. This fraction can be clarified further by a freeze/thaw andcentrifugation cycle without any loss of prothrombin convertingactivity.

Preparation, Isolation, and Purification of Ultra-Pure Thrombin

Assay for Thrombin Activity

Thrombin clotting activity was determined using a modified NIH method.The solutions used consisted of: a) Imidazole buffer, stock, (IBS) madeby dissolving 1.72 g imidazole in 90 mL of 0.1N hydrochloric acid andthen made to 100 mL with distilled water (final pH should be about 7.2).b) PEG/IBS solution made by diluting IBS, 58.8 mL; sodium chloride, 9.0g; and polyethylene glycol (PEG, molecular weight 8000), 5 g to 1000 mLwith water. Normal human plasma was used as a source of fibrinogen andwas diluted with 0.154M sodium chloride (1:1) prior to use. NIH thrombinwas used as a standard and was diluted with polyethylene glycol(8000)/imidazole buffered saline (PEG/IBS) to give 5 U/mL. Clottingassay was performed using a fibrometer. Diluted plasma (200 μL) wasincubated at 37° C. for 3 minutes, then standard thrombin (100 μL) wasadded and the clotting time (in seconds) was recorded (14 to 15seconds). Thrombin-containing unknown sample was diluted with PEG/IBS togive clotting time values higher and lower than the standard clottingtime value by about 5 seconds. Enzyme activity was calculated asfollows: ##EQU1## A: high dilution factor of unknown thrombin sample B:low dilution factor of unknown thrombin sample

C: average clotting time of standard thrombin

D: average clotting time of low dilution of unknown thrombin sample

E: average clotting time of high dilution of unknown thrombin sample

g: units of standard thrombin in test mixture divided by volume ofstandard thrombin in test mixture (0.5/0.1)

Enzyme activity is expressed as Units/mL (up to 8000 U/mL) and asUnits/mg protein (up to 10,000 U/mg protein).

Purified or partially purified bovine plasma prothrombin is reacted withpurified thromboplastin in the presence of 10-40 mM calcium chloridesolution at a temperature between 10° and 25° C. for 15-45 minutes asdescribed under Example B, "Isolation of Ultra-Pure Thrombin". Theamount of thromboplastin activity is two to three times that ofprothrombin at pH 6.5-7.0. The thrombin produced by this reaction isfurther purified as follows. The resulting protein suspension iscentrifuged (12K RPM) in a refrigerated centrifuge (2°-10° C.) toseparate insoluble nonactive proteins. The supernatant is loaded on aweak anion-exchange column (DEAE-Sepharose®, DEAE-Sephadex®, DE-52®).The column is eluted with 25-50 mM phosphate buffer (pH 6.5) containing0.1M sodium chloride (2°-10° C.). The eluant is monitored at 280 nM.Fractions with high UV absorbance are further checked for thrombinclotting activity (using a fibrometer). These pooled fractionscontaining thrombin are turbid and are clarified by freezing thesuspension overnight, followed by thawing (<25° C.) and centrifugationor filtration. The thrombin-containing pool is loaded on acation-exchange column (CM-Sepharose®, CM-Sephadex®) and eluted using asalt gradient (0.1M to 1M sodium chloride in 25-50 mM phosphate buffer,pH 6.5 ).

The eluant is monitored at 280 nM and the fractions containing thrombinactivity (as determined by the fibrometer) are pooled (FIG. 1). Thefractions containing ultra-pure thrombin are water clear and may haveabout 8000 U/mL of activity. The purity of ultra-pure thrombin isdetermined by reversed-phase HPLC, polyacrylamide gel electrophoresis,and isoelectric focusing.

Protein content of the thrombin fraction was determined using theBradford assay (Bradford, M., Anal. Biochem., 72:248, 1976) and theprotein reagent made by BioRad (Richmond, Calif.). Specific activity ofthe preparation was calculated by dividing the amount of enzyme unitsper unit volume into the amount of proteins per same unit volume.

The exceptionally high specific activity of thrombin made by the presentinvention is attributed to the following:

1. The use of a freshly harvested prothrombin yields a thrombin productof high specific activity. Also, inactive thrombin can co-elute withactive thrombin and this can result in decreased specific activity ofthe final product. Therefore, all the isolation steps need to be carriedout at a temperature between about 2° to 7° C. Furthermore, solutionswere not allowed to stand, even refrigerated, for extended periods oftime prior to use. Freezing at about -10° to -20° C. was adequate toprotect the products.

2. The use of highly purified thromboplastin as described in the presentinvention diminishes the extent of contaminants or the presence ofnonspecific proteins in the final thrombin preparation, hence increasingthe specific activity.

The Thrombin Preparations

The preparations made in accordance with the invention must contain, ina liquid medium, ultra-pure thrombin, and one or more buffers. They maycontain saline, and other substances conventionally employed in proteinpreparations.

While the term "preparations" is employed, it should be noted thatApplicants contemplate all types of formulations in which thrombin, insubstantially solubilized form, is present in combination with one ormore glycols and buffers.

When a liquid formulation is made, it is generally preferred that thesolvent(s) or other diluent(s) employed have a suitable miscibility withthrombin such that production standards, e.g., uniformity of thrombinconcentration from batch to batch, can be readily met.

The thrombin employed is an ultra-pure thrombin obtained by the processof the present invention.

This thrombin solution is, if desired, then mixed with glycerolcontaining either acetate buffer or phosphate buffer and saline, inorder to prepare a stabilized solution.

Thrombin is known to be soluble in physiological saline--i.e., asolution containing about 0.9% NaCl in water. However, other salinesolutions are contemplated as useful herein. Furthermore, thereplacement of all or part of the NaCl in such solutions with one ormore other suitable salts is contemplated.

Water is a preferred medium for the preparations of the invention.However, the use of one or more other diluents which do not adverselyaffect the solubility and/or stability of thrombin in the subjectpreparations is desirable.

One such diluent is glycerol. Other useful polyols include mannitol,sorbitol, sucrose, glucose, and the like. Mixtures are operable.Glycerol is highly preferred.

The glycerol or other polyol ingredient(s) will be employed at a totalconcentration of from about 10 to about 40 wt. %, preferably 20 to 30wt. % based on total composition weight.

Unless stated otherwise, all quantities recited are weight percentagesbased on total compositions weight.

Suitable buffer systems are those whose aqueous solutions will maintainpH of the final thrombin solution between about 5.0 and about 8.0, witha preferred pH range of about 5.5 to about 6.5. It is highly preferredthat when a phosphate buffer is used the final pH of the preparation beabout 6.0 to about 6.5 and when an acetate buffer is used, the final pHbe about 5.0.

pH measurements are made using an ordinary pH meter with a combinationelectrode.

Useful buffer systems include acetate, phosphate, succinate,bicarbonate, imidazole, TRIS, and the zwitterionic buffers described byN. E. Good and S. Izawa, in Methods in Enzymol, 24, Part B, 53 (1972);and W. F. Ferguson, K. I. Braunschweiger, W. R. Braunschweiger, J. R.Smith, J. McCormick, C. C. Wasmann, N. P. Jarvis, D. H. Bell, and N. E.Good in Anal. Biochem 104, 300 (1980). These disclosures are herebyincorporated by reference.

Suitable reagents for use in the instant buffer systems include MES,ACES, BES, MOPS, TES, HEPES, and the like. Phosphate should only be usedwhen calcium ion is absent or in the presence of EDTA. Mixtures of suchreagents can be employed. If mixed buffers are used, the final pH shouldbe suitably adjusted.

Buffers containing phosphate ion and acetate ions are preferred.Mixtures are operable.

The buffers will be present in the buffer solution, along with waterand/or other suitable diluent(s) at total concentrations of about 0.01Mto about 0.2M, preferably about 0.025M to about 0.10M.

The use of various other conventional additives, e.g., antioxidants,colorants, surfactants, and the like, is also contemplated. Glutathionemay be employed as an optional ingredient. Amino acids may be employedas optional ingredients, but their presence must not be in suchquantities as to interfere with the stabilizing action of the polyol andbuffer components on the purified thrombin. In general, it is preferredthat they be used in only minute quantities at concentrations of 0.5% orless, if at all.

Hemostats

Hemostatic materials, such as GELFOAM®, SURGICEL®, and AVICEL®, andcollagen, which are presently used alone or in combination with thrombinpowder or thrombin in saline, can be effectively used with thestabilized thrombin formulations of the present invention using avariety of techniques. Preferably, the stabilized solution is absorbedonto the hemostatic agent and the pad is freeze-dried and packaged in asterile manner.

Antimicrobial or antibiotic agents can also be incorporated into suchpads, especially for use on burn patients, where prevention of infectionis critical.

One type of bandage suitable in the preparation of coagulants inaccordance with the invention is set forth in U.S. Pat. No. 4,363,319,the disclosure of which is hereby incorporated by reference.

The following is illustrative of the preparation of an ultra-purethrombin solution.

EXAMPLE

A. Isolation of Thromboplastin:

100 g Veal lung was homogenized in 200 mL, 25 mM sodium phosphatebuffer, pH 6.5, containing 0.5% polysorbate 80 and centrifuged at 12KRPM for 20 minutes at 5° C. The supernatant was collected and the pHadjusted to 5.72 using 10% acetic acid, and let stand in therefrigerator for 1 hour. The mixture was centrifuged for 20 minutes asabove and the supernatant collected. 160 mL of the supernatant wasloaded on a CM-Sepharose® column (30×20.5 cm), which was saturated with25 mM sodium phosphate buffer, pH 6.5, and eluted using the same buffer.Fractions of 230 drops were collected (about 13 mL) and the opticaldensity at 280 nM of the fractions was measured.

    ______________________________________                                        Tube No.      OD at 280 nM                                                    ______________________________________                                         1 clear      0.0                                                              2 clear      0.0                                                              3 clear      0.0                                                              4 opalescent 1.369                                                            5 opalescent 3.751                                                            6 opalescent 3.828                                                            7 opalescent 0.451                                                            8 yellowish  3.427                                                            9 yellowish  3.993                                                           10 reddish    3.513                                                           11 reddish    4.060                                                           12 reddish    4.071                                                           13 reddish    4.055                                                           14 reddish    4.073                                                           15 reddish    3.944                                                           16 reddish    3.723                                                           ______________________________________                                         Pool 1 fractions 4-6                                                          Pool 2 fractions 7-9                                                     

The pools were assayed for thromboplastin activity by convertingprothrombin to thrombin as follows:

    ______________________________________                                        Mix         prothrombin 1.0       mL                                                      saline      0.5       mL                                                      pool fract. 0.1       mL                                                      CaCl.sub.2 (0.3 M)                                                                        100       μL                                       ______________________________________                                    

at 25° C. for 25 minutes, centrifuged at 5° C. at 13K for 10 minutes andassayed for thrombin clotting activity using a fibrometer.

    ______________________________________                                        Fraction    Clotting time (seconds)                                           ______________________________________                                        Pool 1      10.9                                                              Pool 2       9.9                                                              ______________________________________                                    

The two pools contained significant thromboplastin (prothrombinconverting) activity. The first pool was used for the conversion ofprothrombin to thrombin since it has less color. The second pool,however, could be utilized also.

B. Isolation of Ultra-Pure Thrombin

The conversion mix was made of:

    ______________________________________                                        Prothrombin            97 mL                                                  saline                 40 mL                                                  CaCl.sub.2 (0.3 M)     10 mL                                                  Thromboplastin         15 mL                                                  ______________________________________                                         (Pool 1)                                                                 

incubated at 25° C. for 30 minutes and centrifuged at 13K for 20 minutesat 5° C., and collected the supernatant.

C. DEAE-Sepharose Column Chromatography

Equilibrated the column (30×2.5 cm) with 25 mM sodium phosphate bufferpH 6.5 containing 0.02% sodium azide. Sodium azide was used as abacteriostatic agent, however, this agent cannot be used during theisolation of thromboplastin since it causes browning of the redhemoglobin. Other common bacteriostatic agents can be substituted andare preferred. These include phenols and substituted phenols,chlorobutobenzyl alcohol, benzalkonium chloride, benzethonium chloride,thimerosal, and phenylmercuric nitrate.

Loaded 115 mL of the converted preparation on the column and elutedusing the same buffer containing 0.1M sodium chloride. Collectedfractions of 230 drops and assayed for thrombin clotting activity.

Fractions 7-48 showed significant thrombin clotting activity; they werepooled (510 mL) and the material appeared turbid. The pooled materialwas kept in a plastic bag and frozen overnight. The material was thawed,centrifuged at 13K for 20 minutes at 5° C. and the supernatantcollected.

D. CM-Sepharose Column Chromatography

Equilibrated the column (30×2.5 cm) with 25 mM sodium phosphate buffer,pH 6.5 containing 0.02% sodium azide and 0.1M sodium chloride.

Loaded 493 mL of the previous supernatant from the DEAE-sepharose columnstep.

Eluted the column with a salt gradient made of 225 mL of the buffercontaining 0.02% sodium azide and 0.1M sodium chloride and 225 mL buffercontaining 0.02% sodium azide and 1.0M sodium chloride. Fractions werecollected as described above and assayed for thrombin clotting activityusing a fibrometer.

Fractions 15-21 contained significant activity and were pooled (volumeof 90 mL). The pooled material was then assayed for thrombin activityand showed clotting activity of 8213 U/mL.

This pooled fraction was also assayed for protein content using theBradford method and Bio-Rad protein assay kit. The pooled materialcontained 0.82 mg/mL protein.

Final specific activity=8213/0.82=10015 U/mg protein.

We claim:
 1. An ultra-pure, clear, colorless bovine thrombin solutionhaving a specific activity of from about 8000 to about 11,000 units permg protein as determined by a modified NIH-method.
 2. The thrombinsolution of claim 1 having a specific activity of about 9000 Units permg protein.
 3. A thrombin solution of claim 2 prepared by reachingprothrombin with purified thromboplastin and treating the resultingthrombin after centrifugation by eluting the supernatant through ananion-exchange agarose column; freezing then thawing up to about 25° C.the desired eluant fraction; centrifuging, and eluting the supernatantthrough a cation-exchange agarose column with a 0.1M to 1M sodiumchloride salt gradient in a phosphate buffer.